Although most in vitro studies of CNS development, transplantation, regeneration, neuropathology or electrophysiology require only small amounts of neural tissue, huge amounts of neural tissue are wasted in these studies because of the difficulty of culturing any but the youngest fetal neurons. Improved methods for tissue dissociation, tissue culture and cryopreservation of more mature neurons can be developed. This will reduce the numbers of animals required for in vitro neurobiology in several ways: 1. Cryopreservation allows efficient use of neurons from entire rodent litters or from larger (or less readily available) fetuses or neonates. 2. Cryopreservation (allowing transport of CNS tissues) and culture of older neurons allows investigators (even distantly located) to use many different CNS tissues from the same animal, despite variations in relative maturity of the neurons. This encourages collaboration using larger or less readily available species (such as rabbit, primate or human), which may better represent human neurochemistry, neurophysiology and neuropathology. 3. Neonates routinely culled from large litters could provide sufficient CNS tissues for many studies. 4. Cryopreservation could provide commercial sources of CNS tissues for schools and research laboratories which require only small amounts of tissues. Dissociation with milder enzyme solutions, lower sodium and calcium levels and anesthetics or ion blockers to minimize activity during processing will be tested, as will culture media containing growth- promoting hormones and neurotrophic factors to increase survival.